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McLatchie, Alex P; Burrell-Saward, Hollie; Myburgh, Elmarie; Lewis, Michael D; Ward, Theresa H;Mottram, Jeremy C; Croft, Simon L; Kelly, John M; Taylor, Martin C; (2013) Highly sensitive in vivoimaging of Trypanosoma brucei expressing "red-shifted" luciferase. PLoS neglected tropical diseases,7 (11). e2571-. ISSN 1935-2727 DOI: https://doi.org/10.1371/journal.pntd.0002571
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Highly Sensitive In Vivo Imaging of Trypanosoma bruceiExpressing ‘‘Red-Shifted’’ LuciferaseAlex P. McLatchie1, Hollie Burrell-Saward1, Elmarie Myburgh2, Michael D. Lewis1, Theresa H. Ward1,
Jeremy C. Mottram2, Simon L. Croft1, John M. Kelly1*, Martin C. Taylor1
1 Faculty of Infectious and Tropical Diseases, London School of Hygiene and Tropical Medicine, London, United Kingdom, 2 Wellcome Trust Centre for Molecular
Parasitology, Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow, United Kingdom
Abstract
Background: Human African trypanosomiasis is caused by infection with parasites of the Trypanosoma brucei speciescomplex, and threatens over 70 million people in sub-Saharan Africa. Development of new drugs is hampered by thelimitations of current rodent models, particularly for stage II infections, which occur once parasites have accessed the CNS.Bioluminescence imaging of pathogens expressing firefly luciferase (emission maximum 562 nm) has been adopted in anumber of in vivo models of disease to monitor dissemination, drug-treatment and the role of immune responses. However,lack of sensitivity in detecting deep tissue bioluminescence at wavelengths below 600 nm has restricted the wide-spreaduse of in vivo imaging to investigate infections with T. brucei and other trypanosomatids.
Methodology/Principal findings: Here, we report a system that allows the detection of fewer than 100 bioluminescent T.brucei parasites in a murine model. As a reporter, we used a codon-optimised red-shifted Photinus pyralis luciferase(PpyRE9H) with a peak emission of 617 nm. Maximal expression was obtained following targeted integration of the gene,flanked by an upstream 59-variant surface glycoprotein untranslated region (UTR) and a downstream 39-tubulin UTR, into a T.brucei ribosomal DNA locus. Expression was stable in the absence of selective drug for at least 3 months and was notassociated with detectable phenotypic changes. Parasite dissemination and drug efficacy could be monitored in real time,and brain infections were readily detectable. The level of sensitivity in vivo was significantly greater than achievable with ayellow firefly luciferase reporter.
Conclusions/Significance: The optimised bioluminescent reporter line described here will significantly enhance theapplication of in vivo imaging to study stage II African trypanosomiasis in murine models. The greatly increased sensitivityprovides a new framework for investigating host-parasite relationships, particularly in the context of CNS infections. Itshould be ideally suited to drug evaluation programmes.
Citation: McLatchie AP, Burrell-Saward H, Myburgh E, Lewis MD, Ward TH, et al. (2013) Highly Sensitive In Vivo Imaging of Trypanosoma brucei Expressing ‘‘Red-Shifted’’ Luciferase. PLoS Negl Trop Dis 7(11): e2571. doi:10.1371/journal.pntd.0002571
Editor: Jayne Raper, New York University School of Medicine, United States of America
Received July 19, 2013; Accepted October 21, 2013; Published November 21, 2013
Copyright: � 2013 McLatchie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was funded by grants from the Bill and Belinda Gates Foundation (www.gatesfoundation.org) (Global Health Grant no. OPPGH5337) and theWellcome Trust (www.wellcome.ac.uk) (grant number 092573). The Wellcome Trust Centre for Molecular Parasitology is supported by core funding from theWellcome Trust (grant number 085349). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of themanuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
African sleeping sickness, or human African trypanosomiasis
(HAT), currently infects around 10,000 people per year and
threatens the lives of a further 70 million people living in 36
countries of sub-Saharan Africa [1]. Infections of domestic
livestock are also a major economic problem throughout the
region. HAT is caused by protozoan parasites of the Trypanosoma
brucei species complex which are transmitted to mammalian hosts
by the tsetse fly during a blood meal. There are two sub-species of
human infective parasite. Trypanosoma brucei gambiense, which is
responsible for 90% of clinical cases, causes a chronic form of the
disease. T. brucei rhodesiense, which is primarily zoonotic, causes an
acute form of HAT in eastern Africa. The sub-species Trypanosoma
brucei brucei is non-human infectious, but is a pathogen of domestic
animals.
HAT has two distinct stages. The first is characterised by
recurrent fever and malaise as the parasite replicates in the blood
and lymph. The second begins once parasites penetrate the blood
brain barrier and access the central nervous system (CNS). This
can occur weeks (T.b. rhodesiense) or years (T.b. gambiense) after the
initial infection. Without chemotherapeutic intervention, this leads
rapidly to neurological impairment, coma, and death. The drugs
currently used to treat HAT can be difficult to administer, are
associated with a range of side effects, and treatment failures are
frequently reported [2,3]. The front line drugs, pentamidine and
suramin, were developed over 50 years ago and are only useful
against the haemo-lymphatic stage of disease. Pentamidine is the
drug of choice against T.b. gambiense, while suramin is only
employed to treat T.b. rhodesiense infections. Melarsoprol and
eflornithine, are both used to treat the second stage of infection
following CNS invasion, but both have significant disadvantages.
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Melarsoprol is directly associated with encephalopathic syndrome,
which arises in 5–10% of cases and has a mortality rate of 50–70%
[4,5]. Eflornithine causes less severe side effects, but it is expensive
and difficult to administer, requiring four infusions of
400 mg kg21 each day over a 7 to 14 day period, and is not
used against T.b. rhodesiense. More recently, a nifurtimox and
eflornithine combination therapy (NECT) has been introduced
that is more effective against late stage T.b. gambiense infections
than eflornithine monotherapy. With increasing accounts of
clinical relapse and the growing likelihood of drug resistance,
there is an urgent need to develop novel drugs that are safe,
effective during stage II CNS disease, and easy to administer in the
field.
A current murine model of chronic stage II trypanosomiasis
utilises the pleomorphic T.b. brucei GVR35 strain [6]. However, in
vivo evaluation of compounds for trypanocidal activity using this
system is both labour and animal intensive and requires 180 days
post-treatment to completely assess efficacy. Non-invasive whole
body bioluminescence imaging can be utilized as an alternative
approach in a variety of in vivo disease models to accelerate drug
Author Summary
Parasites of the Trypanosoma brucei species complex are thecausative agents of human African trypanosomiasis. There isan urgent need for new drugs to treat this debilitating andpotentially fatal infection, especially in its late stage, whenparasites have entered the central nervous system. Factorswhich hamper drug development include the limitations ofthe current murine models for stage II disease. In vivobioluminescence imaging is a non-invasive technique thatcan be used to monitor infections in real time and is apowerful new approach for studying drug effectiveness.However, application of this imaging technology totrypanosome infections has been restricted because of lackof sensitivity. In this paper, we have taken a major step toresolve this problem. The enhanced sensitivity in infectedmice is based on the high level expression in trypanosomesof a ‘‘red-shifted’’ luciferase variant that greatly improvesbioluminescence detection in deep tissue. The system whichwe have developed should be a widely applicable tool forproviding new insights into the infection biology of T. brucei.
Figure 1. Schematic of pTb-AMluc constructs containing luciferase reporter genes. We used a modular format for the construction of the pTb-AMlucseries of vectors that were designed to target the ‘‘red-shifted’’ luciferase genes PpyRE9H and PpyRE-TS (red) to the rDNA locus of T.b. brucei. The ribosomal locustargeting fragments (green), and VSG, GPEET2 procyclin and tubulin flanking sequences (blue) were derived from parasite DNA by PCR as outlined in Materialsand Methods and Supplementary Table 1. IR refers to the intergenic region that the contains processing signal(s) at the 59-end of the puromycin N-acetyltransferase (PAC) (purple) and the 39-end of the REH9 luciferase genes. Constructs were linearised by SacI/KpnI digestion prior to transfection.doi:10.1371/journal.pntd.0002571.g001
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discovery and development [7–9]. A particular advantage of
bioluminescence imaging is that the detected signal can be directly
correlated with pathogen load. This provides a simple, non-
invasive way of quantifying the effect of therapeutic intervention in
real time. The use of a stage II trypanosomiasis murine model that
incorporates easily detectable bioluminescent parasites, would
greatly streamline the drug testing process, particularly when
assessing activity against late stage brain infections.
One limitation of bioluminescence imaging results from the loss
of optical resolution of parasites situated in deep tissue. This is
caused by the absorption and scatter of light which decreases the
detectable signal by up to ten fold per centimetre of tissue [10].
Haemoglobin is primarily responsible for this phenomenon in
mammals. However, because haemoglobin absorbs light mostly in
the visible blue-green spectrum, this loss of sensitivity can be
reduced by using red-shifted bioluminescent reporters that emit
light above 600 nm [11,12]. Previous reports of in vivo imaging of
T. brucei infections were based on expression of Renilla luciferase,
which emits light in the 480 nm range [13,14], and firefly
luciferase with emission at 562 nm [15].
Figure 2. Isolation of T.b. brucei clones expressing high level bioluminescence. (A) The bioluminescent signal (RLU) detected from T.b.brucei s427 clones transfected with pTb-AMluc constructs in which the PpyRE9H gene was flanked by 59-VSG and 39-tubulin, 59-GPEET2 procyclin and39-tubulin, or 59-GPEET2 procyclin and 39-actin UTRs (Figure 1). Luciferase assays were carried out on 106 cells (Materials and Methods), with readingstaken using a SpectraMax M3 Microplate Reader. The data show that the 59-VSG/39-tubulin UTR combination in construct pTb-AMluc-v gives thehighest luciferase signal in vitro. Clones 1–4 (indicated) were used in assessment of growth (Figure S1). (B) The bioluminescent signal detected fromT.b. brucei GVR35 clones transfected with construct pTb-AMluc-v. Clone VSL2 (indicated) was chosen for in vivo studies.doi:10.1371/journal.pntd.0002571.g002
Figure 3. Limit of detection in vitro. (A) Images of two 96-well microtitre plates containing dilutions of T.b. brucei GVR35 clone VSL2 and a non-transformed control (WT). Each plate was imaged using an IVIS Lumina (Perkin Elmer) with 1 minute exposure and medium binning. Both cell lineswere serially diluted from 16106 to 16103 (upper plate) and from 16103 to 16102 parasites ml21 (cell numbers shown above each plate). 100 VSL2parasites could be clearly visualised. Note that the imaging software automatically adjusts the heat-map scale to account for the intensity of the wellcontaining the highest number of parasites. (B) In vitro linear regression plots generated from both plates. Each point corresponds tobioluminescence represented by the total flux recorded from a single well. In both cases, linear regression analysis shows a very strong positivecorrelation between bioluminescence and parasite number (R2.0.99). The graphs show readings from duplicate wells. In the upper graph, duplicatevalues were extremely close and are not individually distinguishable. In the lower graph, duplicates are shown as red squares and blue triangles, andthe dotted line indicates the background in blank wells (green triangles), plus two standard deviations.doi:10.1371/journal.pntd.0002571.g003
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Figure 4. Limit of detection in vivo. (A) Six sets of three BALB/c mice were inoculated i.p. with either 20, 100, 500, 5000, or 50000 bloodstreamform T.b. brucei GVR35 clone VSL2. An additional set of three mice was inoculated with 30000 non-transformed parasites (WT). All of the IVIS Lumina
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Here, we report the development and optimisation of T.b. brucei
Lister 427 and T.b. brucei GVR35 reporter cell lines that
constitutively express a red-shifted mutant firefly luciferase with
an emission maximum of 617 nm. These parasites are detectable
in deep tissue at numbers considerably lower than previously
reported and will have numerous in vivo imaging applications.
Materials and Methods
Ethics statementBALB/c and C57BL/6N studies were carried out under UK
Home Office regulations (Project licence PPL 70/6997). The CD-
1 mouse work was performed with the approval of the University
of Glasgow Ethics Committee (Project licence PPL 60/4442).
Parasite culture and transfectionT.b. brucei Lister 427, MITat 1.2 (clone 221a) bloodstream forms
were cultured and maintained in vitro with HMI-9 medium [16],
supplemented with 10% foetal bovine serum (FBS) at 37uC in 5%
CO2. T.b. brucei GVR35 bloodstream forms were cultured and
maintained in HMI-9 medium supplemented with 20% FBS, 20%
serum plus (Sigma) and 0.1% methyl cellulose. Both strains were
electroporated with the Amaxa Nucleofector II, using program X-
001 and human T-cell nucleofector buffer, and 10 mg (Lister 427),
or 30 mg (GVR35) of linearised construct DNA. Transgenic
bloodstream forms were selected and maintained with 1 mg ml21
puromycin.
Construct design and developmentTo generate the parental integration construct pTb-AMluc
(Figure 1), a 250 bp fragment derived from the T.b. brucei
ribosomal DNA (rDNA) promoter was amplified from genomic
DNA using a forward primer that introduced a 59-SacI site and
a reverse primer that introduced 39-MluI and NotI sites (all
primers used are described in Table S1). In addition, a 563 bp
fragment from the rDNA non-transcribed spacer region was
amplified using a forward primer that introduced a 59-ApaI site
and a reverse primer that introduced a 39-KpnI site. The
amplicons were digested with SacI/NotI and ApaI/KpnI,
respectively, and ligated sequentially either side of a puromycin
resistance cassette in a pBluescript backbone (Figure 1). To test
59-untranslated regions (UTR), fragments derived from T.b.
brucei VSG221 (198 bp) and T.b. brucei GPEET2 procyclin (89 bp)
genes were amplified using forward primers that introduced 59-
NotI sites and reverse primers that introduced 39-XhoI sites.
Each amplicon was NotI/XhoI digested and ligated into
separate pTb-AMluc vectors. To test 39-UTR regions, frag-
ments from the corresponding regions of T.b. brucei VSG221
(198 bp) and actin A (289 bp), were amplified using forward
primers that introduced BamHI sites and reverse primers that
introduced HindIII sites. Each 39-UTR amplicon was BamHI/
HindIII digested and ligated into the corresponding vectors.
The red-shifted luciferase genes PpyRE9H and PpyRE-TS [12]
were amplified with a 59-forward primer that introduced an
XhoI site and a reverse primer that introduced a 39-BamHI
site. Each reporter gene fragment was XhoI/BamHI digested
and ligated into separate backbones, to create the final
constructs (Figure 1). Prior to transfection, vectors were
linearised by SacI/KpnI digestion.
In vitro luciferase assays, parasite growth curves andanalysis of construct stability
Luciferase assays were performed with a kit according to the
manufacturer’s protocol (Promega). Prior to each assay, cells were
grown to mid-logarithmic phase and 56106 cells were pelleted by
centrifugation. The cells were lysed by resuspension in 50 ml of
CCLR buffer (Promega). 10 ml of this was added to 100 ml of
luciferase substrate. End point or spectral luminescent readings
were taken using a SpectraMax M3 Microplate Reader (Molecular
Devices GmbH). Growth was monitored by counting each cloned
cell line on consecutive days over a 12 day period, followed by
daily dilution back to 16105 parasites ml21. Construct stability
was tested by culturing each clone in the presence or absence of
1 mg ml21 puromycin and assaying luciferase activity weekly over
a period of three months.
Mice infection, imaging and welfareFemale BALB/c, CD1 and C57BL/6N mice were purchased
from Charles River (MA., USA) and housed in individually
ventilated cages. For infection studies, mice (18–20 g) were
inoculated intraperitoneally (i.p.) with parasites in 200 ml HMI9
medium. For imaging, mice were inoculated i.p. with 200 ml D-
luciferin (15 mg ml21 in Mg/Ca-free Dulbecco’s modified PBS)
(Perkin Elmer), and 10 minutes later anaesthetised with 2.5%
isofluorane. Light emission was recorded using Lumina or
Spectrum in vivo imaging systems (IVIS) (Perkin Elmer). Exposure
times varied between 1 second and 5 minutes, depending on
signal intensity. Peripheral parasitemia was scored by counting
Giemsa-stained smears of tail blood. To clear peripheral
parasitemia, mice were treated with a single 40 mg kg21 oral or
i.p. dose of berenil (1,3-Bis(49-amidinophenyl)triazene) and imaged
between 4 and 7 days later. Mouse brains were excised after
perfusion for ex-vivo imaging. Briefly, mice were exsanguinated
under terminal anaesthesia, then underwent vascular perfusion via
the hepatic portal vein with 10 ml PBS. Successful perfusion was
assessed by blanching of the liver. The whole brains were then
excised and imaged in the Lumina IVIS. To enhance the signal
and avoid desiccation, approximately 100 ml D-luciferin
(15 mg ml21 in Mg/Ca-free Dulbecco’s modified PBS) was
pipetted onto the surface of each brain 5–10 minutes prior to
imaging [15,17]. In accordance with local animal welfare
regulations, infected mice were euthanised when they displayed
immobility, hind-leg paralysis, or 20% weight loss.
Results
Construct optimisationIn trypanosomes, protein coding genes can be transcribed by
RNA polymerase I (Pol-I), leading to high levels of expression [18].
We therefore designed integration vectors where the biolumines-
cent reporter genes would be targeted to the rDNA loci, under the
control of a Pol-I dependent rDNA promoter (Figure 1).
Constructs containing the red-shifted luciferase genes PpyRE-TS
(thermostable) and its derivative PpyRE9H (humanised codon
images were acquired using large binning, 5 minute exposures, 15 minutes after infection and 10 minutes after administration of luciferin(150 mg kg21). It was possible to visualise as few as 100 parasites in the intra-peritoneal space. (B) A dose response curve generated from the in vivolimit of detection data. Mean abdominal bioluminescence was recorded from each group. Linear regression analysis shows a very strong positivecorrelation between bioluminescence and parasite inoculum (R2.0.99). Dotted line indicates mean bioluminescence plus two standard deviations,from mice infected with wild type parasites.doi:10.1371/journal.pntd.0002571.g004
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Figure 5. Monitoring the course of infection. (A) Bioluminescence (total flux) recorded from two BALB/c mice inoculated i.p. with 36104 T.b.brucei GVR 35 (clone VSL2) vs peripheral blood parasitemia recorded over the course of 36 days. Following infection, the bioluminescence signal
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usage) [12], flanked by the 59-GPEET2 procyclin splice site and 39-
tubulin UTR sequences, were first used to transfect bloodstream
form T.b. brucei Lister 427. This is a monomorphic strain in which
transformants can be generated in only 5–7 days. Parasite clones
expressing PpyRE9H were found to display much higher
luciferase activities than those expressing PpyRE-TS (data not
shown). A series of constructs was then generated to identify the
combination of 59- and 39-UTR regions which facilitated the
optimal level of PpyRE9H expression (Figure 1). Analysis of
transformed clones revealed a range of luciferase activities in each
case (Figure 2A). Interestingly, replacement of the 39-tubulin
intergenic sequence with the corresponding sequence from the
actin A gene, resulted in a general reduction of expression levels in
transfected trypanosomes. The highest expression levels were
found in parasites transfected with a construct (pTb-AMluc-v)
containing the 59-VSG and 39-tubulin UTR pairing. We next
transfected the pleomorphic T.b. brucei GVR35 strain with the
pTb-AMluc-v construct. Again, we observed a wide range of
expression levels (Figure 2B). One clone (VSL2) expressed
luciferase activity that was almost 10-fold greater than the highest
expressing Lister 427 clone.
To establish if integration and expression of the reporter gene
affected parasite growth, GVR35 and Lister 427 clones were
followed in culture. There were no significant differences in the
growth rate of transgenic and wild type cell lines (Figure S1). To
ensure that reporter gene expression was stable over time, cell lines
were sub-cultured in the absence of selective drug pressure for up
to 3 months. This time span equates to more than 100 generations
in the pleomorphic GVR35 cell line and 300 generations in the
monomorphic 427 cell line. No significant changes in biolumi-
nescence were detected (Figure S2A and B).
Limit of detection in vitro and in vivoWe assessed if there was a direct correlation between
bioluminescence and cell number by conducting luciferase assays
on serial dilutions of the VSL2 parasite clone (Figure 3). Linear
regression analysis showed a strong positive correlation between
the level of bioluminescence and the number of live cells
(R2 = .0.99). We also sought to determine the limit of detection
achievable with the Lumina IVIS. Serial dilutions of the VSL2
clone in a 96-well microtitre plate format were imaged after the
addition of luciferin (Materials and Methods). 100 parasites could
be readily visualised (Figure 3A).
To determine the limit of detection in vivo, BALB/c mice were
inoculated i.p. with a range of parasite loads and imaged following
injection with luciferin (Figure 4A). Using a maximum exposure
setting, it was possible to visualise as few as 100 parasites in the
intra-peritoneal space. This level of sensitivity was between two
and four orders of magnitude greater than has been reported
elsewhere with trypanosomatid parasites [7,13,14,19–21]. There
was also a strong positive correlation between bioluminescence
and parasite inoculum when the total flux was recorded from
individual mice (Figure 4B). This suggests the feasibility of
assessing in real time the extent of parasite burden over several
orders of magnitude directly from bioluminescence during the
course of an infection, even when parasites are undetectable by
microscopic analysis of peripheral blood.
Monitoring the course of infection and the effect of drugtreatment
The course of infection in BALB/c mice was assessed
following the inoculation i.p. with 36104 VSL2 T.b. brucei
GVR35 parasites (Materials and Methods). This experimental
model of chronic stage II trypanosomiasis is characterised by
CNS infection and death after approximately 35 days, in the
absence of drug treatment. The mice were imaged one day after
infection and then at subsequent time points (Figure 5). At each
time point, blood smears were also taken from the tail to
quantify peripheral parasitemia. Bioluminescence imaging al-
lowed the development of infection to be visualised throughout
the entire experiment, whereas parasites only became detectable
in peripheral blood 14–18 days post-infection (Figure 5A). The
subsequent transient decrease in bloodstream parasitemia
observed after this time point presumably results from anti-
body-mediated killing and antigenic variation within the parasite
population. During this apparent clearance of infection, as
judged by microscopic examination of blood, parasitemia was
readily detectable by in vivo imaging (day 24, Figure 5A). Brain
infections could be tentatively identified within 7 days of
inoculation, particularly when mice were imaged from the
dorsal perspective. After 32 days, the mice were treated with
berenil, a drug which can resolve bloodstream, but not CNS
infections [22]. As expected, there was a dramatic effect, with
complete clearance of parasitemia, as judged by microscopic
examination of blood smears (Figure 5A, B). With biolumines-
cence imaging however, it was clear that parasites had not been
eradicated, with specific foci of infection in the cranial area.
Examination of excised brains from other infected mice (day 33)
revealed clear evidence of wide-spread infection, with distinct,
highly intense bioluminescence (Figure 5C). Interestingly,
bioluminescence was observable 4 days post-treatment in the
region of the mouth and nose (Fig. 5B). This signal could be
indicative of parasite survival in the nasal associated lymphoid
tissue or result from trypanosomes which had spread from the
CNS via the lymphatics into the plate region. In the absence of
drug treatment, the mean survival time (Materials and Methods)
for BALB/c mice infected with the bioluminescent VSL2 clone
was 34.461.2 days (n = 14 mice).
To assess the increased sensitivity achievable with the ‘‘red-
shifted’’ luciferase, as compared to the wild type reporter gene, we
carried out parallel infections of CD-1 mice. One set was infected
with VSL2 T.b. brucei GVR35 trypanosomes and the second with
parasites expressing a yellow firefly luciferase (LUC2) [15]
integrated at a rDNA locus (Figure 6). Images taken 7 days and
21 days post-infection clearly show the enhanced bioluminescent
signal achievable with the ‘‘red-shifted’’ reporter. Similarly, when
fluctuated, peaking at 261011 photons/sec/cm2 on day 18, and remaining above 561010 photons/sec/cm2 until drug intervention. After treatmentwith berenil on day 32 (Materials and Methods), total flux and peripheral parasitemia fell rapidly. (B) Ventral and dorsal in vivo imaging of both mice(Materials and Methods) revealed the rapid growth and dissemination of parasites during the course of infection. Dorsal imaging suggests that asearly as day 7, parasites can be detected in the head of both mice. After berenil treatment, the bioluminescent signal was rapidly cleared from theperiphery and by day 36 was only weakly detectable. However, there was a strong focal signal localised to the head, particularly apparent whenviewed from the dorsal perspective. These mice were sacrificed in accordance with animal welfare regulations. (C) Brains were removed from othermice 33 days post-infection and imaged after perfusion (Materials and Methods). All brains from infected animals showed a clear bioluminescentsignal.doi:10.1371/journal.pntd.0002571.g005
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Figure 6. Comparison of firefly luciferase and the ‘‘red-shifted’’ variant as reporters in vivo. CD-1 mice were inoculated with 36104
bloodstream form trypanosomes expressing yellow firefly luciferase (GVR-LUC2) and the red-shifted variant (GVR35-VSL2). Ventral body (upper) anddorsal head (lower) images of mice were taken after 7 and 21 days using the IVIS Spectrum. Bioluminescence (total flux) and peripheral bloodstream
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parasitemia values are shown. The mice were then treated i.p. with berenil (Materials and Methods) and imaged 7 days later.doi:10.1371/journal.pntd.0002571.g006
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mice were then treated with berenil, residual brain infections were
much more readily detectable in mice infected with the VSL-2
parasites.
We also examined the sensitivity of imaging when applied to
C57BL/6N mice, the most commonly used strain for the
generation of transgenic lines. In this background we observed
that the level of bloodstream parasitemia was lower than in the
BALB/c strain. In addition, there appeared to be some quenching
of the signal, presumably due to the black fur of this mouse strain.
Nevertheless, the course of infection could be easily monitored
with the bioluminescence system and it appeared to follow a
similar pattern to that in BALB/c mice (compare flux traces in
Figure 5A and Figure 7A). Distinct signals appeared in the head
region between day 14 and day 24 (Figure 7) corresponding to the
CNS infection seen in the BALB/c mice.
Discussion
The major goal of the work described here was to improve the
tools available for in vivo bioluminescence imaging of trypanosome
infections, as an aid to drug development programmes and studies
on disease pathology. We addressed two major parameters that in
combination have led to a greatly increased sensitivity of detection.
First, we optimised stable high level expression of the reporter gene
in transgenic parasites, and second, we used a variant luciferase
protein that had been modified to enhance deep tissue detection in
vivo [12].
The expression vector used in this study was designed to target
the multi-copy rDNA loci with the bioluminescent reporter gene
under the control of a ribosomal promoter (Figure 1). Previous
work had shown that this strong promoter can facilitate high level
expression of protein coding genes in trypanosomes [18]. In
addition, we hypothesised that in situ transcription termination
signals at the targeted locus would prevent the aberrant expression
of downstream genes. We chose to avoid integration at a
polycistronic Pol-II locus, such as the tubulin gene array, in case
perturbation of local gene expression had phenotypic consequenc-
es. There are eighteen rRNA arrays in the diploid T. brucei
genome, spread over several chromosomes [23]. When transfec-
tants were examined for luciferase activity, we found significant
differences between clones derived from the same electroporation
(Figure 2). This may reflect differing levels of epigenetic control,
either repression or de-repression, at the targeted rDNA loci.
These observations emphasise the need to examine a number of
clones for luciferase activity each time a new parasite isolate is
transfected with this class of construct. As post-transcriptional
control of gene expression in trypanosomatids is of particular
importance [24], we also tested various sets of 59- and 39-UTR
sequences in the context of rDNA integration, to determine the
best combination for expression. As shown (Figure 2), the highest
levels of expression were associated with construct pTb-AMluc-v,
which contain sequences from 59-VSG and 39-tubulin untranslated
regions. Motifs in these UTR regions of trypanosomatid genes can
have a major influence on transcript processing and stability, or
translational efficiency.
Most in vivo bioluminescence imaging studies previously
published have used luciferase reporter proteins which emit light
in the 480–560 nm range [7–9,13–15]. However, this wavelength
coincides with the region of the spectrum where light absorbance
by haemoglobin is maximal, with a concomitant quenching of the
signal when parasites are localised in deep tissue. Here, we used a
thermostable luciferase variant from the North American firefly
Photinus pyralis, which had been mutated to generate light with a
peak emission wavelength of 617 nm [12]. In addition, the gene
(PpyRE9H) had been altered to conform to human codon bias,
which is similar to that of T.b. brucei [25], and modified further to
remove non-coding repeats, local hairpins and cryptic splice sites
[12]. When mice were infected with transformed bloodstream
forms expressing PpyRE9H in the context of the integrated pTb-
AMluc-v vector, it was possible to detect as few as 100
trypanosomes when concentrated in the peritoneum, with a direct
correlation between parasite load and the total bioluminescent flux
(Figure 4). Parasites in brain might be less readily detected due to
light absorption and scattering by the skull. The pattern of
infection and pathology mirrored that obtained with non-
transfected T.b. brucei GVR35, an experimental model for stage
II trypanosomiasis (Figure 5). Consistent with this, when
transformed bloodstream parasites were monitored in vitro, there
was no evidence of an effect on growth and reporter gene
expression was stable for at least 3 months in the absence of
selective pressure (Figure S1, S2).
The level of imaging sensitivity reported here significantly
surpasses that which has been reported previously with trypano-
somatid parasites [7,13,14,19–21]. It permits infections to be
followed in real time, in a non-invasive manner, even when
peripheral bloodstream parasitaemia is sub-patent. The ability to
image such low numbers of trypanosomes will find widespread use
for monitoring chronic CNS infections where the level of parasite
burden may be low. In addition, it will allow the visualisation of
tissue-specific clearance of infection after drug treatment, without
the need for sacrificing mice at each stage of the process (Figure 5B,
as example). An additional benefit of this bioluminescent parasite
line is that it is easily detectable in C57BL/6 mice (Figure 7), the
background used in most transgenic experiments. With this non-
invasive imaging technology, it will therefore be feasible to assess
the contributions of different genes to host-parasite interactions,
parasite dissemination and CNS infection, using genetically
modified bioluminescent trypanosomes in combination with
transgenic mouse strains. The genes encoding the ‘‘red-shifted’’
luciferase variants used in this study [12] should also be
transferable to T. cruzi and Leishmania to enhance the applicability
of imaging technology to experimental infections with these
related pathogens.
Supporting Information
Figure S1 Cumulative growth of cultured bloodstream form T.
brucei expressing the ‘‘humanized’’ thermostable red-shifted
luciferase (Ppy RE9H). Upper graph; T. brucei s427 and four
independent bioluminescent clones (see Figure 2). Lower graph; T.
brucei GVR35 and the highly expressing bioluminescent line VSL2
used in most of the imaging experiments. Parasite growth was
monitored as outlined in the Materials and Methods.
(PDF)
Figure 7. Monitoring the course of infection in C57BL/6N mice. (A) Bioluminescence (total flux) and peripheral blood parasitemia in miceinoculated i.p. with 36104 T.b. brucei GVR35 (clone VSL2). Following infection, the bioluminescence signal fluctuated, but remained above 1010
photons/sec/cm2 until termination of the experiment. Mouse 1, which developed a heavier infection, died at day 25. (B) Ventral and dorsal in vivoimaging (Materials and Methods) revealed the rapid growth and dissemination of parasites during the course of infection. Parasites can be detectedin the heads of both mice by day 24.doi:10.1371/journal.pntd.0002571.g007
Enhanced In Vivo Imaging of Trypanosoma brucei
PLOS Neglected Tropical Diseases | www.plosntds.org 11 November 2013 | Volume 7 | Issue 11 | e2571
Figure S2 Stability of luciferase expression in the absence of
selective pressure. (A) The Ppy RE9H luciferase signal of a T. brucei
s427 clone parasite maintained in the presence or absence of
1 mg ml21 of puromycin. (B) The Ppy RE9H luciferase activity
expressed by a T. brucei GVR35 clone. No significant decrease in
bioluminescence was observed during this time, showing stable
inheritance of luciferase expression. Each experiment was
performed in triplicate and values shown are the means 6 SD.
(PDF)
Table S1 Primer sequences.
(PDF)
Acknowledgments
We thank Bruce Branchini and colleagues (Department of Chemistry,
Connecticut College) for the kind gift of genes encoding ‘‘red-shifted’’
luciferases, Ryan Ritchie (University of Glasgow) for technical assistance,
and Michael Barrett (University of Glasgow) for input to the design of
experiments and discussion of data.
Author Contributions
Conceived and designed the experiments: APM EM THW JCM SLC
JMK MCT. Performed the experiments: APM EM HBS MDL MCT.
Analyzed the data: APM EM JMK MCT. Wrote the paper: APM JMK
MCT. Provided technical advice: THW JCM SLC. Commented on
manuscript: EM HBS MDL THW JCM SLC. Supervised project
direction: JCM SLC JMK MCT.
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Enhanced In Vivo Imaging of Trypanosoma brucei
PLOS Neglected Tropical Diseases | www.plosntds.org 12 November 2013 | Volume 7 | Issue 11 | e2571
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